Technology to detect very low quantities of nucleic acids associated with various infectious agents (including viruses, bacteria, fungus and protozoa) has advanced rapidly over the last ten years. Such technology includes highly sophisticated hybridization assays using probes in conjunction with amplification techniques such as polymerase chain reaction (PCR) and ligase amplification methods. Researchers have readily recognized the value of such technology to detect diseases and genetic features in human or animal test specimens. The use of probes and primers in such technology is based upon the concept of complementarity, that is the bonding of two strands of a nucleic acid by hydrogen bonds between complementary nucleotides (also known as nucleotide pairs).
PCR is a significant advance in the art to allow detection of yew small quantities of a targeted nucleic acid. The details of PCR are described, for example, in U.S. Pat. No. 4,683,195 (Mullis et al), U.S. Pat. No. 4,683,202 (Mullis), and U.S. Pat. No. 4,965,188 (Mullis et al) and by Mullis et al, Methods of Enzymology, 155, pp. 335-350 (1987), although there is a rapidly expanding volume of literature in this field.
Once the target nucleic acid has been amplified, it can be detected using various techniques. Various devices have been designed for hybridization assays whereby the target nucleic acid is insolubilized prior to detection using a capture probe. For example, nitrocellulose filters and other planar, solid supports have been used to immobilize capture probes for this purpose. One technique used to attach probes to the support is to merely dry them down. More recently, they have been attached to polymeric particles which have been fused to the support (see U.S. Pat. No. 5,173,260 of Zander et al). Still again, such polymeric particles having capture probes can be adhered to supports using polymeric adhesives and various treatments of the supports, as described for example, in EP-A-0 530 357. None of these references, however, describes how such capture probes can be used for semi-quantitative detection of a target nucleic acid.
Various analytical assays, including those used with amplification methods, are based on the detection of a detectable species, such as a dye, fluorescent substance, or radioisotope on the surfaces of small (micrometer or smaller) particles. The results of such assays can be evaluated with sophisticated optical devices to provide truly quantitative results, as in the evaluation of signals generated in clinical chemistry test devices (such as EKTACHEM.TM. test slides). Assay results can also be read visually or with simpler equipment (such as a photometer) to merely provide a positive or negative result.
It would be highly desirable to be able to obtain a quick, semi-quantitative evaluation in an assay using simple equipment such as a photometer. By "semi-quantitative" it is meant that the detection allows for estimating that the result is within several specific ranges of absolute values. For example, it would be desirable to determine whether a target nucleic acid is present in a small, intermediate or large concentration, or simply not at all. Presently, this capability is available only using sophisticated and expensive analytical equipment and tedious procedures which require multiple dilutions of patient samples. However, most laboratories or doctors' offices do not have such equipment available.